(a) Field of the Invention
The invention relates to DNA fragments encoding Abi-type proteins, determined herein respectively as AbiTi and AbiTii, which confers resistance to either naturally or artificially transformed bacteria against bacteriophages. The DNA fragments can be stably or transiently introduced, via a recombinant vector, into host fermentation bacteria. The invention encompasses isolated DNA fragments, recombinant vectors and transformed bacterial strains comprising the two AbiTi and AbiTii fragments.
(b) Description of Prior Art
Lactococcus lactis is a Gram-positive lactic acid bacterium used to acidify milk during the manufacture of cheese, sour cream and buttermilk. Lytic bacteriophages that attack L. lactis can slow down or completely inhibit the milk fermentation and are therefore responsible for substantial economic losses in the dairy industry. Naturally found in raw milk, they resist the pasteurization process, and can spread rapidly within a production plant as they have a short latent period and a relatively large outburst. Lactococcal phages are classified into twelve genetically distinct species. Three of them (936, P335 and c2) are the main cause of troubles since members of these species are regularly isolated worldwide.
Phages are always found in dairy environments and, therefore, the strategy is to control rather than eradicate them. Phage control relies on a variety of practical approaches ranging from adapted factory design, improved sanitation, adequate ventilation, processing changes, starter medium, culture rotation, and the use of phage-resistant starters.
Various techniques are available to protect the sensitive strains by transforming them into phage-resistant derivatives. The development of bacteriophage-insensitive mutant strains (BIM) was formerly the ideal method. A BIM is a spontaneous phage-resistant derivative that survives a long-term exposure to lytic phages, most likely as a result of a change in the phage receptor. Although this technique has the convenience of simplicity and rapidity, this natural evolutionary process is now rarely used for commercial Lactococcus strains due to substantial drawbacks including narrow phage-specificity, reversion of the resistance phenotype, and altered cell growth.
Decades of progress in the genetics of Lactococcus have established a solid basis for constructing another type of phage-resistant starters. Some L. lactis strains were found to naturally possess plasmids coding for anti-phage systems that can be transferred into industrial strains. These anti-phage mechanisms are divided into four groups according to the moment of their action in the phage lytic cycle. Adsorption and DNA ejection inhibition mechanisms block respectively the first and second steps of the lytic cycle. Restriction/modification (R/M) systems cut the viral DNA once it has entered the cell. Finally, abortive infection (Abi) systems can act at any step between DNA ejection and cell lysis but their precise modes of action remain unknown. The resistance conferred by these natural lactococcal plasmids is often broad and effective against most phages of the same species. The discovery of these anti-phage barriers had a significant impact, as resistance strategies became based on phage species rather than on phage isolates (BIMS).
Phage resistant starter cultures have been successfully used for more than 15 years in industrial large-scale fermentation and many of anti-phage systems are subjected to limited exploitation. Over the same period, new phages have continued to emerge and the biodiversity of the phage population has never been so apparent. Unfortunately, some of these emerging phages are insensitive to the resistance mechanisms introduced into the industrial strains. Thus, there is a need for novel anti-phage systems.
The U.S. Pat. No. 5,994,118 describes a DNA encoding phage resistance protein. There is disclosed in this patent an Abi900 protein isolated from a natural 11 kb plasmid of Lactococcus lactis. The protein confers resistance to some bacteriophage infections.
The U.S. Pat. No. 4,931,396 describes a plasmid pTR2030 and derivatives thereof containing genetic determinants which confer phage resistance to group N streptococci. The plasmid has also a conjugal transfer phenotype upon expression in group N streptococci. 
The U.S. Pat. No. 5,928,688 describes a DNA encoding phage resistance protein which aborts infection by the phage, designated as AbiE. The DNA, which is contained in a Lactococcus lactis deposited as NRRL-B-21443, is incorporated into a bacterium to encode the AbiE and provide phage resistance.
Antibacterial or antiviral systems are always offset by the emergence of resistant strains. Natural phage-resistant systems are not immuned to this evolutionary process.
It would be still highly desirable to be provided with modified or new phage-resistance factors, capable of conferring resistance to different bacteria used in fermentation processes.